Genotypic and Phenotypic Characteristics Associated with Biofilm Formation by Human Clinical Escherichia coli Isolates of Different Pathotypes

Abstract

Bacterial biofilm formation is a widespread phenomenon and a complex process requiring a set of genes facilitating the initial adhesion, maturation, and production of the extracellular polymeric matrix and subsequent dispersal of bacteria. Most studies on Escherichia coli biofilm formation have investigated nonpathogenic E. coli K-12 strains. Due to the extensive focus on laboratory strains in most studies, there is poor information regarding biofilm formation by pathogenic E. coli isolates. In this study, we genotypically and phenotypically characterized 187 human clinical E. coli isolates representing various pathotypes (e.g., uropathogenic, enteropathogenic, and enteroaggregative E. coli). We investigated the presence of biofilm-associated genes ("genotype") and phenotypically analyzed the isolates for motility and curli and cellulose production ("phenotype"). We developed a new screening method to examine the in vitro biofilm formation ability. In summary, we found a high prevalence of biofilm-associated genes. However, we could not detect a biofilm-associated gene or specific phenotype correlating with the biofilm formation ability. In contrast, we did identify an association of increased biofilm formation with a specific E. coli pathotype. Enteroaggregative E. coli (EAEC) was found to exhibit the highest capacity for biofilm formation. Using our image-based technology for the screening of biofilm formation, we demonstrated the characteristic biofilm formation pattern of EAEC, consisting of thick bacterial aggregates. In summary, our results highlight the fact that biofilm-promoting factors shown to be critical for biofilm formation in nonpathogenic strains do not reflect their impact in clinical isolates and that the ability of biofilm formation is a defined characteristic of EAEC.IMPORTANCE Bacterial biofilms are ubiquitous and consist of sessile bacterial cells surrounded by a self-produced extracellular polymeric matrix. They cause chronic and device-related infections due to their high resistance to antibiotics and the host immune system. In nonpathogenic Escherichia coli, cell surface components playing a pivotal role in biofilm formation are well known. In contrast, there is poor information for their role in biofilm formation of pathogenic isolates. Our study provides insights into the correlation of biofilm-associated genes or specific phenotypes with the biofilm formation ability of commensal and pathogenic E. coli Additionally, we describe a newly developed method enabling qualitative biofilm analysis by automated image analysis, which is beneficial for high-throughput screenings. Our results help to establish a better understanding of E. coli biofilm formation.

Keywords: Escherichia coli; VideoScan; biofilm formation; pathotypes.

FIG 1

Colony morphologies and colors of different E. coli isolates on a Congo red agar plate. Bacteria were grown at 37°C for 24 h on LB agar plates without salt containing Congo red dye. E. coli Nissle 1917 developed a rough and dry colony morphology with red color based on the binding of Congo red dye (rdar morphotype). E. coli isolate 5212 (EPEC) exhibited a brown and shinier color but also a dry and rough colony morphology (bdar morphotype). The isolate E. coli 5644 showed smooth and white-colored colonies (saw morphotype).

FIG 2

Fluorescence of different E. coli isolates on a calcofluor agar plate. Bacteria were grown at 37°C for 24 h on LB agar plates containing calcofluor. Cellulose-producing bacteria showed a fluorescent colony morphology (E. coli Nissle 1917). E. coli isolate 5644 (EAEC) exhibited no cellulose production.

FIG 3

Swimming motility of E. coli on a soft agar plate. Bacteria were stabbed in the center of the agar plate and incubated at 37°C for 16 h. A positive motility was indicated by diffused growth around the point of inoculation (E. coli MG1655 F′Tet). Growth restricted along the stab point indicated a nonmotile isolate (E. coli 5148).

FIG 4

Biofilm formation of E. coli isolates in four different culture media analyzed with the VideoScan technology. Dot plots showing the relative fluorescence intensities ([relFIs] AU) of SYTO 9-stained bacteria, which were examined for biofilm formation in four different media (LB, TSB, BHI, and M63). Each dot represents the median value of the relFI. The biofilm assays were performed three times with triplicates in each assay. The dotted lines mark the cutoffs for biofilm formation in each medium. Open circles indicate no biofilm and solid circles indicate biofilm formation. EAEC isolates exhibited the highest relFI in enriched media (LB, TSB, and BHI). The relFIs differed significantly between the pathotypes (0.95 confidence level). To show these differences, pathotypes were grouped. Pathotypes in group A are significantly different from pathotypes in group B. For example, in LB medium, the pathotypes AFEC, CAEC, HFEC, SAEC, and UPEC (group A) exhibit significantly higher relFIs than EPEC and ETEC (group C) and significantly lower relFIs than EAEC (group B). The groups for pathotypes were created separately for each medium.

FIG 5

Overview image of E. coli biofilms in a 96-well plate. Merged single-well fluorescence images show a 96-well plate with SYTO 9-stained biofilms. From left to right: beads that were used as an internal reference and five EAEC isolates (5270, 5271, 5272, 5275, and 5276) are shown. Each isolate was examined for biofilm formation in four media (LB, TSB, BHI, and M63) in triplicate (box).

FIG 6

Single-well overview images of E. coli isolates showing different strengths of biofilm formation. Fluorescence images were captured after 48 h of biofilm formation in respective media and staining with SYTO 9. (A) The positive control E. coli MG1655 F′Tet ΔtraD in LB medium exhibiting strong biofilm formation (relFI, 0.4398). (B) One HFEC isolate in M63 medium displaying an even biofilm in the center and a much more pronounced biofilm along the edge of the well (relFI, 0.1102). (C) One UPEC isolate in M63 medium forming a flat and evenly distributed biofilm in the well (relFI, 0.0369). (D) One EAEC isolate in BHI medium showing the characteristic biofilm consisting of thick bacterial aggregates (relFI, 0.2763).